The disclosed methods relate to assays for detecting enzymatic activity. In particular, the disclosed methods relate to the use of thiol-reactive reagents for detecting enzyme activity.
Many enzymes are known to utilize phosphate as a signaling molecule. These include kinases, phosphatases, and enzymes associated with G-protein coupled receptor complexes (GPCRs). Protein kinases are one of the most widely studied classes of enzymes. They have been estimated to represent approximately 1.7% of the human genome, and over 500 kinases have been identified in the human “Kinome.” Many protein kinases have been implicated in hyperproliferative diseases (e.g., cancer), and as such, interest is focused on understanding the function of these enzymes and on identifying modulators of their activity. With the initial identification of kinase-targeted drugs, there has been renewed interest in pursuing protein kinases as drug targets. A number of kinase inhibitors are in various stages of clinical trials. As such, there is tremendous interest in the broad study of ligand-enzyme interactions with respect to kinases, and there is a general need for better tools for studying these interactions.
Commonly used kinase assays are either fixed-time (i.e., assaying data at a single data point) or continuous (i.e., assaying data at multiple data points). Fixed time kinase assays typically employ indirect detection mechanisms, such as monitoring binding of a phosphorylated product to an immobilized metal ion or antibody. Such assays are not ideal, in that they use multiple reagents and employ indirect measurements. Furthermore, typical fixed-time kinase assays require the presence of the kinase's substrate that is phosphorylated. Another disadvantage of all fixed time assays is that because they provide single time-point measurements, they can produce artifactual measurements that could have been diagnosed by monitoring a continuous rate of reaction over the course of the assay.
Continuous assays provide multiple time point measurement to define an enzymatic rate. Commonly, continuous assays involve an enzyme-coupling reaction. In one common enzyme-coupling system, the kinase reaction is coupled to a pyruvate kinase/lactate dehydrogenase reaction (“PK/LDH”) and one of the ultimate reaction products of the PK/LDH reaction is used to monitor kinase activity (i.e., NAD+). In this system, the assayed kinase converts ATP to ADP. Pyruvate kinase then utilizes ADP to generate pyruvate from phosphoenol pyruvate. Finally, lactate dehydrogenase converts pyruvate to lactate and concurrently converts NADH to NAD+. As such, the decrease in concentration of NADH may be monitored over time based on the absorbance of NADH at λ=340 nm and correlated with kinase activity. This system may not be ideal in that it involves three coupled reactions. Further, NADH has a low extinction coefficient (ε340 nm=6.22 mM−1cm−1). In addition, the PK/LDH assay involves monitoring a decrease in signal rather than an increase in signal, which limits the dynamic range and makes the assay more difficult to optimize.
Therefore, although kinase assays are presently available, there is a need for a continuous assay that monitors an increasing signal, and permits assay of kinases even when their natural substrate is unknown, as is common in a functional genomics project. Assays that involve direct detection of ADP, or an analog thereof, are potentially universal assays for kinases, and would not require that the natural substrate be present, because most kinases will slowly hydrolyze ATP even in the absence of their natural substrate (i.e., commonly called the “ATPase” or hydrolysis reaction).
GPCRs (G-protein coupled receptors) play an important role in communicating signals from the outside to the inside of cells. The external signal for these receptors may include light, hormones, growth factors, and various ligands that bind to the GPCR. An agonist signal activates exchange of GTP for GDP bound to the Gα a subunit of the GPCR-complex, which stimulates release of the Gβγ subunits, permitting downstream activation of other proteins. This activated state is only transient though, because the bound GTP is slowly hydrolyzed to GDP by the intrinsic GTPase activity of Gα, and the Gα/GDP complex then rebinds to the Gβγ subunits to reform the inactive state of the GPCR. Furthermore, there are regulators of G-protein signaling (RGS) that can act as GTPase activating proteins (GAPs), and activate the GTPase activity of the Gα subunit. Given the important role of GPCRs in cell biology, as well as their prevalence as drug targets, there is a need for improved assays of GPCR activity, and for methods to quantitate the effect of antagonists—which could serve as potential drug leads. For example, Seifert et al. (J. of Pharmacology and Experimental Therapeutics (2003) 305, 1104-1115) reported the screening and identification of antagonists of the histamine H1 receptor (a GPCR, termed H1R), which could lead to treatments for allergic diseases. The Seifert et al. assay involved monitoring GTPase activity of the Gα subunit of H1R by observing the release of radioactive phosphate from [γ-32P]GTP, analogous to commonly used kinase assays using [γ-32P]-ATP. There is a need for better assay that may be used to monitor the activation state of GPCRs including continuous assays.